JP2022509535A - Cell-free RNA library preparation - Google Patents

Abstract

A variety of cDNA libraries derived from cell-free mRNA and methods for preparing them are provided. Libraries can be prepared by extracting RNA from body fluids such as serum or plasma, separating RNA from contaminants, synthesizing cDNA by reverse transcriptase, and concentrating protein-encoding nucleotide sequences. .. The library can contain a large number of transcripts from solid tissue. cf-RNA can be measured by qPCR, sequencing or other suitable method.

Description

Cross-reference This application claims the benefit of US Provisional Application No. 62 / 752,533 filed October 30, 2018, which provisional application is incorporated herein by reference in its entirety.

Various markers can be used to detect various conditions. However, many of these conditions can affect different tissues. Detecting markers of these conditions in the circulation, for example in blood samples, is not always helpful in identifying which tissue is affected. For example, a genetic marker of inflammation can indicate an inflammatory response somewhere in the body, but it may not be known which tissue is causing the response, such as the liver, kidneys, lungs or joints. Tissue-specific tests, such as biopsies, are often invasive and therefore carry the risk of infection and generally do not cover the entire organ or tissue. Imaging techniques such as MRI and CT scans may be used to assess tissue health, but generally only obvious features and changes can be detected. Therefore, these imaging techniques are generally not sensitive enough to capture the early start of a condition or the most recent occurrence of a condition.

Incorporation by Reference All published documents, patents and patent applications referred to herein are to the same extent as if each individual published document, patent or patent application is specifically and individually indicated to be incorporated by reference. Incorporated herein by reference.

Cell-free mRNA provides a potential window to the health, phenotype and developmental programs of various tissues and organs. The present disclosure provides a diverse cell-free mRNA library enriched with non-blood genes and methods for preparing them.

In one aspect, a method of preparing a cf-RNA sample comprising (a) centrifuging the biological sample at 1,600 g to 16,000 g and (b) isolating the RNA from the biological sample. , At least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 selected from the list in Table 1 or selected from Table 10. A method is provided herein in which a low stringency non-blood gene is present in a cf-RNA sample. The biological sample can be a cell-free biological sample and can be serum, plasma, saliva, urine, interstitial fluid, cerebrospinal fluid, semen, vaginal fluid, amniotic fluid, tears, synovial fluid, mucus or lymph. In certain embodiments, the biological sample is serum or plasma.

The method of preparing a cf-RNA sample may include performing size selection or immunoselection on the biological sample prior to isolating the RNA from the biological sample. In some embodiments, the step of performing the size selection comprises centrifuging the biological sample. Centrifugation can be performed for at least 1 minute, at least 10 minutes, 5 minutes to 20 minutes, 10 minutes to 15 minutes, or about 10 minutes. In some embodiments, the biological sample is centrifuged at 10,000 g to 15,000 g. In some embodiments, the biological sample is centrifuged at about 12,000 g. In some embodiments, the step of performing size selection involves filtering the sample.

In some embodiments, the step of isolating RNA from a biological sample comprises isolating the extracellular vesicles, which may be exosomes, from the biological sample, and isolating the RNA from the extracellular vesicles. In some embodiments, the step of isolating RNA from a biological sample comprises isolating the nucleoprotein complex from the biological sample and isolating the RNA from the nucleoprotein complex.

The method of preparing a cf-RNA sample may further include treating the RNA with a deoxyribonuclease. In some embodiments, the deoxyribonuclease is TurboDNase I. In some embodiments, the RNA is treated with a deoxyribonuclease in solution.

In some embodiments, the step of isolating RNA from a biological sample comprises contacting RNA with at least one of an affinity column, a desalting column, or a silica membrane. In a further embodiment, the RNA is contacted with an affinity column, a desalting column, and a silica membrane.

In some embodiments, the method of preparing a cf-RNA sample further comprises the step of enriching at least one protein-encoding nucleotide sequence. In another embodiment, the method of preparing a cf-RNA sample comprises depleting the ribosomal RNA sequence from RNA.

In another embodiment, a method for identifying a cf-RNA molecule, wherein (a) an RNA is isolated from a biological sample, (b) a cDNA library is prepared from the RNA, and (c) a cDNA library is prepared. At least 50, 100, 200 selected from the list in Table 1 in which the biological sample is substantially cell-free and comprises the steps of sequencing and (d) identifying at least one gene in the cDNA library. , 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 non-blood genes or low stringency non-blood genes selected from Table 10 are detected. , Provided herein. In some embodiments, the method of identifying a cf-RNA molecule further comprises aligning a sequence from a cDNA library with the reference genome.

In this method of identifying cf-RNA molecules, in some embodiments, the biological sample is cell-free. In some embodiments, the biological sample is serum, plasma, saliva, urine, interstitial fluid, cerebrospinal fluid, semen, vaginal fluid, amniotic fluid, tears, synovial fluid, mucus or lymph. In other embodiments, the biological sample is serum or plasma.

In some embodiments, the method for identifying the cf-mRNA molecule is at least 1, 5, 10, 20, 50, 100, 200, 300, 400 or 500 tissue-specific genes selected from Table 2, Table. At least 1, 5, 10, 20, 50, 100 or 150 brain-specific genes selected from 6, liver-specific genes at least 1, 5, 10, 20 or 50 selected from Table 7, or Table 8 Identifies genes for liver diagnosis from, or any combination thereof.

In some embodiments, the method of identifying a cf-RNA molecule provided herein comprises the step of identifying a first gene, where the RNA is aligned with the first gene 500, 200, 150. , 100, 50, 25 or less than 15 cf-mRNA polynucleotides.

In some embodiments of the method of identifying cf-mRNA molecules, at least 2, 4, 6, 8 or 10 unique fragments are detected per 100 reads. In some embodiments, at least 2, 4, 6, 8 or 10 protein-encoding genes are detected per 10,000 reads.

In some embodiments, the method of identifying a cf-mRNA molecule further comprises performing size selection or immunoselection on the biological sample prior to isolating the RNA from the biological sample. In some embodiments, the size selection comprises centrifuging the biological sample. The biological sample can be centrifuged at 1,600 g to 16,000 g and centrifuged for at least 1 minute, at least 5 minutes, at least 10 minutes, 5 minutes to 20 minutes, 10 minutes to 15 minutes, or about 10 minutes. .. In some embodiments, the biological sample is centrifuged at 10,000 g to 15,000 g, or about 12,000 g. In another embodiment, the step of performing size selection involves filtering the sample.

In some embodiments of the method of identifying cf-RNA molecules provided herein, the step of isolating RNA from a biomolecule is to isolate extracellular vesicles from a biological sample, and to isolate RNA. Includes isolation from extracellular vesicles. In some embodiments, the extracellular vesicles are exosomes.

In some embodiments, the step of isolating RNA from a biological sample comprises isolating the nucleoprotein complex from the biological sample and isolating the RNA from the nucleoprotein complex. In some embodiments, the method of identifying a cf-RNA molecule is to add an extrinsic RNA polynucleotide containing a first nucleotide sequence to a biological sample, and to detect a cDNA polynucleotide containing a first nucleotide sequence. The first nucleotide sequence of the cDNA polynucleotide comprises timine at each position where the first nucleotide sequence of the RNA polynucleotide contains uracil.

In some embodiments, the method of identifying a cf-RNA molecule further comprises treating the RNA with a deoxyribonuclease. In some embodiments, the deoxyribonuclease is TurboDNase I. In some embodiments, the RNA is in solution when treated with a deoxyribonuclease.

In some embodiments, the step of isolating RNA from a biological sample comprises contacting RNA with at least one of an affinity column, a desalting column, or a silica membrane. In a further embodiment, the RNA is contacted with an affinity column, a desalting column, and a silica membrane.

In some embodiments, preparing a cDNA library from RNA containing a random sequence that can be a random hexanucleotide. In some embodiments, the concentration of random hexanucleotides is at least 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 150 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1000 μM, 1100 μM, 1200 μM, 1300 μM. It is 1400 μM, or 1500 μM.

In some embodiments, preparing a cDNA library from an RNA step that identifies a cf-mRNA molecule comprises forming a single-stranded cDNA. In some embodiments, the method further comprises contacting the RNA with reverse transcriptase to form a single-stranded cDNA. In a further embodiment, the double-stranded cDNA is formed from the single-stranded cDNA. In yet a further embodiment, the single-stranded DNA is contacted with the NEBNext DNA polymerase to form the double-stranded cDNA. In some embodiments, the method further comprises ligating the unique dual index to both ends of the double-stranded cDNA.

In some embodiments, the method of identifying a cf-RNA molecule further comprises the step of enriching at least one protein-encoding nucleotide sequence. Concentration comprises depleting the ribosomal RNA sequence from RNA in some embodiments and depleting the ribosomal RNA sequence from the cDNA library in some embodiments. In some embodiments, the step of enriching at least one protein-encoding nucleotide sequence comprises isolating at least one protein-encoding sequence from RNA or cDNA. In some embodiments, the step of enriching at least one protein-encoding nucleotide sequence comprises hybridizing the entire exome bait to the cDNA. The whole exome bait can be an RNA polynucleotide or a DNA polynucleotide.

Other aspects of the disclosure provided herein are at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 non-blood genes selected from the list in Table 1. Low stringency non-blood genes selected from Table 10; at least 1, 10, 20, 50, 100, 200, 300, 400, 500, selected from the list in Table 1 per 1,000,000 cDNA polynucleotides. 600, 700, 800, 900 or 1000 non-blood genes or low stringency non-blood genes selected from Table 10; at least 5, 10, 20, 50, 100, 200, 300, 400 or selected from Table 2. 500 tissue-specific genes; at least 1, 5, 10, 20, 50 or 100 brain-specific genes selected from Table 6; or at least 1, 5, 10, 20 or 50 livers selected from Table 7. A cf-mRNA sequencing library containing cDNA molecules resulting from a specific gene.

Yet another aspect of the present disclosure is a cf-mRNA sequencing library containing cDNA polynucleotides resulting from at least 2000, 3000, 4000, 5000 or 6000 protein coding genes, at least 8%, 15 of the protein coding genes. % Or 24% is a non-blood gene, cf-mRNA sequencing library.

The novel features of the present disclosure are specifically demonstrated in the appended claims. Subsequent detailed description showing exemplary embodiments in which the principles of the present disclosure are utilized, as well as the accompanying drawings, will provide a better understanding of the features and advantages of the present disclosure.

FIG. 1 shows a flowchart of a cf-mRNA analysis method according to some embodiments of the present disclosure.

2A-2E show plasma centrifugation with a force in the range of 1,900 g to 16,000 g, or filtration of plasma with a membrane having a pore size in the range of 0.2 um to 0.8 um for erythrocytes (FIG. 2A). ), Platelets (FIG. 2B), neutrophils (FIG. 2C), liver (FIG. 2D) and brain (FIG. 2E) depleting cf-mRNA transcripts. Same as above. Same as above. Same as above. Same as above.

3A-3B are graphs showing enrichment of non-blood cf-mRNA transcripts with increasing centrifugal force. Blood transcripts are depleted at a slower rate than non-blood transcripts. FIG. 3A shows the number of copies of blood and non-blood transcripts. The number of transcripts was normalized to 1.0 at 1900 g rotation. FIG. 3B shows the normalized number of blood and non-blood transcripts per million. For each group, the maximum number of transcripts per million was normalized to 1.0. Same as above.

FIG. 4 is a graph showing the number of non-blood genes detected in the cf-mRNA transcript after centrifugation with a force ranging from 1,900 g to 16,000 g.

FIG. 5 is a graph showing cf-RNA yields for three RNA extraction kits determined by qPCR of β-actin cf-mRNA.

FIGS. 6A-6B are on the QIAamp Cycleting Nucleic Acid Kit using an optimized miRNA extraction protocol (FIG. 6A) compared to the manufacturer's standard nucleic acid extraction protocol (FIG. 6B) determined by capillary electrophoresis on a bioanalyzer. FIG. 6 is a graph showing an increase in yield of shorter cf-RNA polynucleotide fragments. Same as above.

FIG. 7 is a graph showing the improvement in cf-RNA yield in the QIAamp Circulating Nucleic Acid kit using the optimized miRNA extraction protocol compared to the QIAamp cfDNA / RNA kit.

FIG. 8 is a graph illustrating that treatment of extracted c-RNA with TurboDNase I in solution, as determined by capillary electrophoresis on a bioanalyzer, eliminated trace impurities in DNA polynucleotides.

FIG. 9 is a graph showing that removal of the inhibitor from the sample increased the apparent yield of cf-RNA, as determined by qPCR of 18S rRNA.

10A-10B show that the OneStep PCR inhibitor removal column (current desalting column) is more than the other column, the Micro-Bio-Spin column (desalting column 1), as determined by capillary electrophoresis on a bioanalyzer. It is a graph which shows that it retains a small amount of RNA. Same as above.

FIG. 11 is a graph showing the reduction of cf-RNA extraction failure in the method (current) described in Example 1 as compared with the old method (past).

FIG. 12 shows that the recovery of cf-RNA by the method described in Example 1 is linear and consistent with an increase in plasma input, as determined by qPCR of β-actin cf-mRNA. It is a graph which shows.

FIG. 13 is a graph quantifying RNA yields with different reverse transcriptases as determined by qPCR of 18S cDNA.

FIG. 14 is a graph showing the conversion of RNA from various amounts of input cf-RNA to cDNA, as determined by qPCR of 18S cDNA.

FIGS. 15A-15B were prepared using Accel-NGS 1S Plus (Swift 1) and Accel-NGS 2S Plus (Swift 2) (FIG. 15A) and standardized and optimized Accel-NGS 2S Plus protocols (FIG. 15B). It is a graph which shows the unique sequence fragment in a cDNA library. Same as above.

16A-16B are graphs showing misdesignated sequences using the unique dual index (FIG. 16A) as compared to the standard index (FIG. 16B).

FIG. 17 shows the abundance of RNA morphology in total cf-RNA (ver1), in rRNA-depleted cf-RNA (Ver2), and during total exosomal capture of mRNA (Ver3), as determined by DNA sequencing. ..

18A-18C are graphs showing the sensitivity of cf-mRNA sequencing using the method described in Example 1 (Wift 1S) compared to the SMARTer kit with rRNA depletion. Detection sensitivity for ERCC standard material (FIG. 18A). Number of genes detected (Fig. 18B). Illustrative determination of detection sensitivity for ERCC standard material (FIG. 18C). ERCC was a standard material spiked into samples from 4 patients (Pt 7171, Pt, 7131, Pt 7139 and Pt 7155). Same as above.

19A-19E are graphs illustrating a comparison between the cf-mRNA library prepared by the method of Example 1 and the cf-mRNA library described by Pan et al .: (FIG. 19A) Number of sequencing reads. , (FIG. 19B) the number of unique fragments detected, (FIG. 19C) the number of protein-encoding genes detected, (FIG. 19D) coverage> 80% of the genes, and (FIG. 19E) the number of detected liver genes. number. Same as above. Same as above.

Provided herein are methods that can utilize precentrifugation to reduce unwanted "blood" transcript contamination from cf-mRNA sequencing data. The methods herein can reduce background noise resulting from blood cell RNA (“blood components”). Such noise can increase sequencing depth requirements and weaken signals from tissue-specific cf-mRNA.

The protocols, methods and kits disclosed herein are 1,500 g to 20,000 g, 1,900 g to 16,000 g, 4,000 g to 16,000 g, 8,000 g to 16,000, 10,000 g and up. 14,000 g, 11,000 g to 13,000 g, 11,000 g to 12,500 g, about 12,000 g, essentially 12,000 g, substantially 12,000 g, or about 12,000 g, smaller or smaller than these It can be consistent with a wide range of centrifugal forces, such as a large range. Part of the range extends to about 12,000 g. Some ranges are within 100g of 12,000g. Some centrifuge protocols are not significantly different from 12,000 g, such as centrifugation at 12,000 g. Some ranges are within 100g of 16,000g. Some centrifuge protocols are not significantly different from 16,000 g, such as centrifugation at 16,000 g. Alternative ranges with low starting points listed above or high ending points listed above are also conceivable. Such a centrifuge protocol is 2.5 times (eg, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6) in the variety of extracted cf-RNA samples for processing. 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2 9.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 40 times, or 4.0 Can contribute to improvement, such as (greater than double) improvement.

The rate of separation of particles due to gravity applied by centrifugation in a suspension generally depends on the particle size and density. Particles that are denser or larger in size generally move at a faster rate and at some point can be separated from less dense or smaller particles. Alternative techniques for separating particles according to their size include, but are not limited to, gel filtration chromatography and filtration with size-selective membranes. All such techniques are within the scope of this disclosure.

Some commercially available extraction protocols may show high sampling failure rates, may extract small amounts of cf-mRNA, and contain many contaminants that can reduce the performance of downstream assay steps. It may not be possible to eliminate it. Such kits and protocols can only extract subpopulations of either smaller cf-mRNA fragments or larger cf-mRNA fragments. Accordingly, provided herein are methods for extracting cf-mRNA from blood that help generate high quality sequencing data that can be rich in biometric information. The method herein may utilize a kit for consistent extraction of cf-mRNA from blood with a low failure rate and improved yield of cf-mRNA. Such yields may retain both smaller and larger cf-mRNA fragments in order to produce amplifyable cf-mRNA.

As disclosed herein, some techniques can improve sample extraction success or RNA library diversity by retaining the eluate from at least one extraction wash step, and as a result, so. Small RNA polynucleotides that are otherwise lost in the eluate of the wash step are retained, contributing to the diversity of the RNA library for processing.

Low levels of DNA contamination can cause errors in quantifying gene expression, while blood contaminants can interfere with the biochemical properties of downstream assays. In addition, commercially available RNA extraction kits have either ignored the step of removing DNA or recommended on-column DNAse treatment, which may not be optimal for robust removal of DNA. For example, in low yield cf-mRNA samples, low contamination levels can contribute to large data misdisplays. Accordingly, provided herein are methods and systems configured to remove contaminants in blood using cf-mRNA wash conditions. Moreover, such methods can eliminate sporadic genomic DNA contamination of cf-mRNA samples.

Alternatively or in combination, the methods and systems disclosed herein may remove contaminants by adding an enzymatic DNAse step to remove DNA contaminants and / or carryover. Some enzymatic and non-enzymatic DNA removal treatments are consistent with the disclosure herein, which often shares the effect of removing DNA from cf-RNA samples. The methods herein can provide a desalted cleanup column that improves sample amplification potential (eg, by removal of inhibitors) and improves cf-mRNA enrichment, diversity and yield. ..

Oligo-dT priming for cDNA synthesis may not be optimal for fragmented and / or degraded mRNA. In particular, the degraded sample may contain fragments lacking the poly A tail, and incomplete reverse transcription may result in a reverse transcriptase lacking the 5'region. Accordingly, some systems, methods and kits consistent with the disclosure herein are oligos containing up to 4, 5, 6, 7, 8, 9, 10 or more than 10 bases, such as pentamers. It may include adding reagents for random priming of reverse transcription, such as those using hexamer, heptamer, octamer, nonamar or decamer. In some embodiments, hexamer may be used to prime reverse transcription.

In addition, some commercial enzymes may inhibit cDNA production due to inhibitors from previous steps, while cDNA quantification for reverse transcriptase uses known RNA inputs. It may present poor quantitative accuracy when doing so.

Systems and methods that can improve the efficiency of RNA-to- cDNA conversion and the accuracy of cf-mRNA quantification are provided herein. The method herein has selected the best reverse transcriptase to yield the highest quality and quantity of cDNA from RNA input, but is relatively high in oligos such as hexamer rather than oligo-dT priming for cDNA synthesis. Concentrations (eg, higher than those recommended for some commercial kits) may be utilized. Oligos, such as random hexamers or oligos of other lengths, can be used in a concentration range consistent with the disclosure herein. For example, max. Concentrations above 1500 μM, at least about or substantially these concentrations, and about or substantially consistent with these ranges are considered. Concentrations within these ranges, such as at least 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 150 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1000 μM, 1100 μM, 1200 μM, 1300 μM, 1400 μM, or 1500 μM. , Or concentrations above 1500 μM are also consistent with the disclosure herein. That is, in some cases, random oligos such as random hexamers can be used at about 200 μM. In some cases, random oligos such as random hexamers can be used at about 500 μM. In some cases, random oligos such as random hexamers can be used at about 1000 μM. In some cases, random oligos such as random hexamers can be used at about 1500 μM. In some cases, random oligos such as random hexamers can be used at about 2000 μM. Fractional concentrations are also possible.

Alternatively, random oligos, such as random hexamers or oligos of other lengths, can be used at higher concentrations than the amounts recommended in the kit. Concentrations such as 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 21x, 22x, 23x, 24x, 25x, 26x, 27x, 28x, 29x, 30x, 31x, 32x, 33x, 34x, 35x, 36x, 37x, 38x, 39x, 40x, 41x, 42x, 43x, 44x, 45x, 46x, 47x, 48x, 49x, Concentrations in the range of 50x, or higher than 50x, are considered for use in the methods, systems and kits herein. In some cases, concentrations ranging from 15x to 40x, 20x to 35x, 25x to 35x, 28x to 32x, or at least 25x, 26x, 27x, for example 30x. , 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, or more than 35-fold concentrations are used.

Implementation of random hexamers with high amounts and specific reverse transcriptase may allow a robust and accurate amount of cDNA to proceed to library preparation. The methods and systems herein utilize an improved cDNA synthesis process to improve sample failures and improve the abundance and robustness of biometric and tissue-specific transcript identification. The library preparation protocol that has been created can be identified. Such methods can reduce the amount of sequencing resources wasted on non-informational reads, such as ribosomal RNA, which may make up> 80% of the transcriptome. Therefore, the methods and systems herein may include total exosomal enrichment to capture only cf-mRNA. It can show an improvement in assay sensitivity for RNA molecule detection. In addition, the methods and systems herein utilize enriched protocols that are not normally used for RNA preparation to obtain custom probes for capturing spike-in transcript cDNA.

In some embodiments, a selected population of cf-mRNA and / or a selected population of cDNA derived from cf-mRNA is used in the brain, liver, lung, bladder, kidney, heart, breast, stomach, intestine. , Colon, gallbladder, pancreas, lung, prostate, ovary, epithelial tissue, connective tissue, nerve tissue or muscle tissue, etc., can be enriched by hybridization to a bait representing a particular organ or tissue. In some embodiments, a bait or disease or condition that distinguishes a selected population of cf-mRNA and / or a selected population of cDNA derived from cf-mRNA between certain organs or tissues. It can be enriched by hybridization to the bait, which is useful for diagnosis or prognosis prediction.

In some examples, the methods provided herein increase the efficiency of converting RNA into a sequenceable cDNA library, for example by selecting a DNA-seq library kit that exhibits improved efficiency. Can be improved. In some cases, to facilitate the application of the reverse transcriptase cf-RNA kit to the sequencing library protocol, the cDNA library is processed with a second-strand synthase or protocol to cf in the sample. -A population of double-stranded cDNA molecules representing RNA can be generated. Double-stranded DNA molecules can then be subjected to analysis or sequencing library generation using protocols that direct DNA library generation rather than RNA or single-stranded DNA library generation. In some cases, compliance with the library generation protocol directed to double-stranded DNA was directed to library generation from cf-RNA or single-stranded reverse transcriptase for downstream analysis. Produces a higher quality library than that produced by the protocol, such as a sequencing library. In certain embodiments, RNA is added to a reverse transcriptase such as Superscript IV prior to the initiation of a double-stranded DNA-directed sequencing library protocol, eg, prior to a second-strand synthetic regimen, including a NEBNext polymerase. Methods that include contacting the samples can be used to process the cf-RNA.

Methods, systems and kits that can reduce misdesignation of sequencing leads to the wrong sample are also provided herein. Methods and systems can minimize the loss of cf-mRNA libraries from stringent cleanup conditions during the enrichment process. Stringent conditions may be required to prevent carryover of indexed primers that may participate in subsequent PCR amplification of the cf-mRNA-derived library. The method may include the use of reagents from IDT technologies and a unique dual index (UDI) to prevent misalignment of sequencing reads. When using the standard index, the sequencing read was misdesignated as a negative control (NTC).

Since the majority of transcripts found in blood can be derived from blood cells, a list of "non-blood" genes that can be detected in blood is provided herein. This list was determined by fusing sample processing (centrifugation rate) with bioinformatics tools to identify "non-blood" and tissue-specific signatures. Non-blood vs. blood transcripts were determined as a function of centrifugation rate. Centrifugation rates in the range of 8,000 g to 16,000 g provided a balance between the number of transcripts and genes detected and the signal-to-noise ratio.

A partial list of genes suitable for the identification of non-blood cf-RNA transcripts in blood includes: Gene ID SEMA3F; HSPB6; MEOX1; CX3CL1; CDKL3; SEMA3G; DCN; IGF1; WWTR1; PHLDB1; NSAI2; CPS1; RAI14; PREX2; KITLG; ELN; BCAR1; ITIH1; LIMCH1; WISP2; CALCRL; EML1; KIF26A; ACSM2B; ADGRF5; GAL; PTPN21; LMCD1; LNX1; RARB; FBRN1; MAP2; NEBL; HOXX9; RAPGEF3; RIMS1; PTPRH; CADPS2; COL16A1; MECOM; MMP2; PIR; EPB41L1; ARHGAP28; NOS1; FXYD3; RAPGEF4; TF; APOH; FMO2; CRTAC1; PALMD; PALM; CARD10; RASL10A; RBFOX2; GALNT16; CCM2L; PLS3; ASB9; GABER; FLT1; ZNF423; NDRG4; CD276; TJP1; PLAT; TUSC3; MET; HOXX5; TSPAN12; SFRP4; MEOX2; RARRES2; GLI3; OGN; LHX6; PTGR1; AMBP; MPDZ; GLIS3; APBA1; ATRNL1; CXCL12; PALD1; CCL2; COL1A1; ART4; MGP; CDCA3; AICDA; TPD52L1; LAMA4; C7; FGF1; LIFR; DPYSL3; HRG; AMOTL2; RBP1; FGF12; EVA1A; EFEMP1; IGFBP5; EFHD1; TPO; SDC1; NR5A2; ADGRL2; MFAP2; KIF17; HSD11B1; PROX1; APOA1; TTR; ELOVL4; FILIP1; PCDH17; ELOVL3; NKX2-3; TEK; KIAA1217; IQSEC3; TBX2; FABP3; SF8; IL13RA2; SLC12A5; PTGIS; POF1B; HIF3A; HIST1H1A; NRN1; SSUH2; MT1G; ID1; F10; RHOJ; AIF1L; MASP1; PTPRB; KDR; RFPL1; A4GALT; KRT17; CPA4; CGNL1; ISLR; RNASE1; SHC2; DOCK6; APOE; APOC1; USHBP1; UNC13A; PXDN; ASS1; GALNT15; PDLIM4; RAMP2; KHDRBS3; RAI2; NR0B2; RHPN2; PPARG; REEP2; BEX1; PDZD2; SPINK5; LYVE1; MRO; MEIS2; CABLES1; APLNR; COL4A2; TBX3; AMHR2; HEY2; PKIB; STAB2; THSD1; EDNRB; LRRC32; SULF1; YAP1; SMAD6; ARHGAP29; TACC2; RBP4; OIT3; AOX1; DUOXA1; GCSH; GATA6; CCDC40; FKBP10; MMEL1; PRDM16; FCN3; TINAGL1; RGS5; CXorf36; AWAT2; FAM13C; ADIRF; ROM1; OOSP2; CLEC1A; ADGRL3; CCDC102B; DOCK1; MAGI1; THRSP; AKR1C2; PTPN14; HSPB8; TMEM178A; SPARCL1; GJA1; PLOD2; UCHL1; ENAH; PDLIM3; JAM2; FGD5; GNA14; KCNMA1; NMNAT2; CCNB2; AFAP1L1; ERG; HPD; SHROOM4; LAD1; C1QC; CIART; FCN2; AZGP1; COX7A1; ADGRL4; SNX7; VCAM1; DDR2; C1orf115; PIGR; RFTN2; FAM84A; NOSTRIN; FABP1; ALB; PRICKLE2; A DAMTS9; APBB2; TM4SF18; EMCN; SPINK1; MYOZ3; BMPER; ZNF704; COL1A2; SOX17; DEFB1; AQP7; KIAA1462; SMCO2; FBN1; LARP6; SPIC; CYYR1; SEMA6B; GGT6; KLK4; ACER1; GSDMA; DNASE1L2; ACOX2; FAM107A; COL3A1; FAM178B; CPLX1; EFNA1; SHE; ANTXR1; ROBO1; CTNND2; TM4SF1; MYRIP; PDE7B; KRT20; MAP6; FGA; FGB; PAH; ARTT2; SYNPO2; AGXT; MUCL1; SNTG2; GXYLT2; SNCG; STOX2; C1QTNF1; CD34; PHLDA3; PODN; SLCO2A1; DES; CLEC14A; C8orf4; C8G; CASKIN2; PTRF; CALML3; PSAPL1; LGALS7B; WSCD1; PIPOX; CDH5; TMEM45A; OR6S1; C1S; BGN; CLEC4G; PYCR1; CTNNA3; FBXL7; WBP5; NR2F2; KRT79; RGS7BP; KRT14; KRTAP23-1; LYPD6; FAM9C; C11orf96; GJA4; NANOS3; PLA2G2A; C15orf52; S100A16; FSIP2; AADACL3; APOD; S100A13; FAM177B; HOXA4; MFAP5; PARVA; TEAD4; SULT1C4; ADH4; HMGN5; ZNF442; ARHGEF15; DMD; C1orf53; SMIM9; SOX18; AWAT1; IFFL2; ERICH4; MT1M; APOC4-APOC2; CFLAR-AS1; PLCL2-AS1; LA16c-395F10. 1; C14orf132; AC046143.3; PPP5D1; RP11-14N7.2; HLA-DQB2; PHGR1; RP1-67K17.3; APOC2; RP11-758P17.3; TDGF1; INMT; GSTA1; ETV5; RP11-148B6.1; ECSCR; RP11-548K23.11; IQCJ-SCHIP1; SHANK3; RP11-116G8.5; CTD-2135J3.4; RP11-923I11.5; RP11-315D16.2; HOXB7; RP11-521L9.1; RP11-680G10. 1; RP11-1260E13.4; GJA5; CTD-2350C19.2; AP000275.65; RP11-452I5.2; APOC4; AC003002.4; AC007193.8; DOC2B; CCL14; PIK3R3; RP5-1042K10.14; MATR3; RP11-717K11.2; CDR1-AS; and AL365273.1.

Methods that can preferentially deplete blood cell cf-mRNA and thereby improve the ability to detect organ-derived cf-mRNA, which may be of higher informative value for diagnosis, Provided herein. Centrifugation rates and times can be optimized tissue-by-tissue to collect appropriate organ-specific transcripts and to separate cf-mRNA fractions from different organ types. Such isolation of "non-blood" organ-specific cf-mRNA from blood may allow the extraction of meaningful biological information.

At best, cf-RNA live by implementation of at least one of the above methods, including methods, systems and kits that utilize a combination of the above methods that uses the majority or all of the methods herein. Improvements or substantial improvements in rally preparation can be achieved. Such improvements are 2.5-fold in RNA library diversity (eg, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8). 1, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3 .1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 40 times, or 4.0 times larger) Improvements, etc. It can be observed by at least one of the increase in library diversity. Increased library diversity may allow the same number of unique genes to be detected in fewer sequencing reads or more unique transcripts to be observed in the same number of sequencing reads. Similarly, in various cases, an increase or substantial increase in non-blood transcripts sequenced in the cf-mRNA library, eg, maximum or at least 10%, 20%, 30%, 40%, 50%, 60. An increase of%, 70%, or greater than 70% can be observed. Some systems, methods or kits can show an increase of, for example, about 50%. Such an increase can be observed prior to or in conjunction with the selective removal of sequences identified as blood-related transcripts. Such an increase can help or facilitate having a reduced volume, reduced sequencing depth, or both reduced volume and sequencing depth compared to some standard protocols. Can be observed in a sample with. In some cases, the sequencing depth may be maxed out or at least 10%, 20%, 30%, 40%, 50%, 60%, 70% without a corresponding reduction in the number of unique transcripts detected. , Or can be reduced by more than 70%. Some systems, methods or kits can show a reduction of, for example, about 50%, and nevertheless can still provide an improvement in the above diversity. In some cases, the sample volume can be reduced by a maximum or at least 10%, 20%, 30%, 33%, 40%, 50%, 60%, 70%, or more than 70%. Some systems, methods or kits show a reduction of, for example, about 33%, despite the improvements in diversity described above.

In some examples, on an absolute scale, methods, systems and kits consistent with the disclosure herein are low in the sequence read library resulting from the analysis of the sample as provided herein. The resolution of the quantity transcript sequence can be increased. At least or at most 900, 800, 700, 600, 500, 400, 300 per sample, measured by sample transcripts, or by internal standard RNA molecules or externally processed molecules assayed simultaneously or independently. , 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15 or 10 molecules are included in the final sequence dataset. You can observe that you are there. That is, in some examples, it can be observed that the final sequence data set contains transcripts present in a total amount, for example 10 to 100 molecules per sample. This can mean an improvement over some other methods.

The biological sample for cf-mRNA production can be any body fluid. Exemplary body fluids include blood, saliva, urine, interstitial fluid, cerebrospinal fluid, semen, vaginal fluid, amniotic fluid, tears, synovial fluid, mucus or lymph. The cells can be removed from the body fluid by centrifugation or other means, such as filtration. In blood, cf-RNA may be associated with proteins, lipids, salts or other components. Some cf-RNA is released from cells into extracellular vesicles such as exosomes. Exosomes can be isolated by methods such as, but not limited to, sedimentation by centrifugation, size exclusion, filtration, equilibrium density gradient centrifugation, immunoisolation, immunodepletion and combinations thereof.

In some embodiments, the methods of the present disclosure allow or allow detection of one or more extracellular RNA transcripts in a biological sample (eg, body fluid). The biological sample can be serum, plasma, saliva, urine, interstitial fluid, cerebrospinal fluid, semen, vaginal fluid, amniotic fluid, tears, synovial fluid, mucus, lymph, or another suitable body fluid. In various embodiments, the method may allow detection of one or more cell-free mRNA molecules derived from non-blood cells in a serum sample. The method may allow the detection of one or more cell-free mRNA molecules derived from non-blood cells in serum samples in addition to hematopoietic transcripts.

The genes detected in cf-mRNA can identify the tissue of origin and / or organ (eg, tissue-specific genes; see Tables 2-7), or are of particular interest for diagnosing diseases or conditions. It can also be (see Tables 8-9). Moreover, the methods provided herein can be sensitive, and thus with a small number of copies of only 10, 15, 25, 50, 100, 150, 200 or 500 in a biological sample (eg, body fluid). Existing extracellular RNA molecules can be detected. RNA molecules can be detected by sequencing, qPCR, ddPCR, microarrays, or any other suitable method.

The methods provided herein can detect and / or measure extracellular RNA molecules present in biological samples (eg, circulating in body fluids). In various embodiments, the method can detect and / or measure acellular mRNA transcripts derived from hematopoietic and / or non-hematopoietic cells (eg, non-blood genes in Table 1 or Table 10). The method detects 1, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more non-blood genes from Table 1 or Table 10. Purified cf-RNA samples that can be and / or measured can be produced. The method is tissue-specific, organ-specific, at least 1, 5, 10, 20, 50, 100, 200, 300, 400 or 500 from cf-RNA extracted from a biological sample, eg, from Tables 2-9. Alternatively, genes important for diagnosis can be measured or detected. The method is to generate a cf-RNA sample from a biological sample capable of detecting RNA molecules present in copies of at most 10, 15, 25, 50, 100, 150, 200 or 500 or less. Can be done.

Also provided herein are methods of detecting at least 10, 20, 30, 50 or 100 non-blood cf-mRNA genes in a biological sample. The method is not limited to these, and (a) the serum or plasma sample is centrifuged at 8,000 g to 16,000 g (or other range as provided herein) for at least 10 minutes and the supernatant. Steps to form, (b) extract RNA from the supernatant, (c) contact RNA with deoxyribonuclease, (d) form cDNA from RNA, (f) prepare a cDNA library from cDNA Steps to, (g) sequence the cDNA library, and / or (h) align the sequence with the reference genome to at least 10, 20, 30, 50 or 100 non-blood cf-mRNA per biological sample. It may include the step of identifying the sequence resulting from the gene.

The method may also include (i) contacting a cDNA library with a bait containing a polynucleotide fragment from at least 10, 20, 30, 50 or 100 genes of interest to enrich the gene to be translated. In some cases, method (d) involves contacting the RNA with reverse transcriptase to form a single-stranded cDNA, and contacting the single-stranded cDNA with a double-stranded synthase to form a double-stranded cDNA. It may include forming cDNA. The method may also include (j) ligating a unique dual index to a cDNA library to form an indexed cDNA library. In some embodiments, the method may include (k) pooling up to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more indexed cDNA libraries. The method may also include (l) performing a massively parallel sequencing on the pooled cDNA library.

In some embodiments, at least 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 genes are detected in a biological sample. In various embodiments, the sequence is aligned with the reference genome to at least 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 non-blood cf- per biological sample. The sequence resulting from the mRNA gene can be identified. The method may further comprise the step of contacting the single-stranded cDNA with a double-stranded synthase to form a double-stranded cDNA. In some cases, (c) can be performed in solution.

In certain embodiments, the method of detecting at least 10, 20, 30, 50 or 100 non-blood cf-mRNA genes in a biological sample is, but is not limited to, (a) a serum or plasma sample. A step of centrifugation or filtering from 900 g to 16,000 g (or other range as provided herein), (b) a step of extracting an RNA sample from the supernatant, (c) an RNA sample with deoxyribonuclease. Contacting, (d) contacting RNA with reverse transcriptase to form single-stranded cDNA, (e) forming double-stranded cDNA from RNA, (f) double-stranded cDNA in the cDNA library Preparation from, (g) contacting a bait containing a polynucleotide fragment with an indexed cDNA library to concentrate the gene to be translated, (h) sequencing the cDNA library, and / or ( i) The sequence may be aligned with the reference genome to identify sequences originating from at least 10, 20, 30, 50 or 100 non-blood cf-mRNA genes per biological sample.

The method may further include (j) adding a unique dual index to the cDNA library to form an indexed cDNA library (eg, by ligation, PCR, etc.). In some embodiments, the method may include (k) pooling up to 10 indexed cDNA libraries. The method may further include (l) performing a massively parallel sequencing on the pooled cDNA library. In some cases, the method may further comprise the step of contacting the single-stranded cDNA with a double-stranded synthase to form a double-stranded cDNA. In some embodiments, (c) can be performed in solution.

Polynucleotide sequences that are "aligned" with a gene generally have about 100% identity to some or all of the sequences in the gene.

As used herein, the singular forms "one (a)", "one (an)" and "the" are plural references unless otherwise expressly indicated by the context. Including the subject. All references to "or" herein are intended to include "and / or" unless otherwise stated.

Although preferred embodiments of the present disclosure are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variants, modifications and substitutions will immediately come to the minds of those skilled in the art without departing from the present disclosure. It should be understood that various alternatives of the embodiments described herein are available in the practice of the present disclosure.

The present application can be better understood by reference to the following non-limiting examples provided as exemplary embodiments of the present application. The following examples are presented to better illustrate the embodiments, but should not be construed as limiting the broad scope of the present application in any way.

(Example 1 method)
The processes used for acellular mRNA (cf-mRNA) analysis include biological sample processing, cf-mRNA extraction, cf-mRNA purification, cDNA synthesis, library preparation, DNA sequencing, and bioinformatics (FIG. 1).

Blood was collected on EDTA vacutainer (BD) for plasma treatment or on a red cap vacutainer (BD) for serum treatment. Blood was incubated for at least 30 minutes at room temperature for serum treatment. After storage at room temperature for less than 2 hours after collection, blood was centrifuged at 1600 g for 10 minutes to obtain plasma or serum (supernatant). The sample can be processed or frozen for storage at −80 ° C. Plasma / serum was centrifuged at 10,000 g to 16,000 g for 10 minutes for a second time to remove residual cells from frozen or fresh samples.

Cell-free RNA was extracted and purified using reagents from the QIAamp Circulating Nucleic Acid Kit (Qiagen Catalog No. 55114). The following conditions were used for up to 1 ml of serum or plasma. The supernatant was transferred to a new tube, mixed with 130 μl Proteinase K, 1.1 ml buffer ACL (without carrier), and 330 μl buffer ATL and incubated at 60 ° C. for 45 minutes. The product was mixed with 3 ml buffer ACB, 1 μl diluted ERCC RNA Spike-In Mix (Life Technologies Catalog No. 4456740), and 2.5 ml cold isopropanol and incubated on ice for 5 minutes. Samples were loaded onto a QiAmp Mini column using a vacuum manifold and then washed with 600 μl buffer ACW1, followed by 750 μl buffer ACW2, and 2 × 750 μl EtOH. The column was dried at 56 ° C. for 10 minutes. RNA was then eluted with 50 μl buffer AVE twice, each for 3 minutes at room temperature, followed by 1 minute centrifugation at 16,000 g. The eluate (approximately 100 μl) was treated with 3 μl of Turbo DNase (Life Technologies Catalog No. AM1907) in 1 × Turbo DNA buffer for 20 minutes at 37 ° C. The reaction was stopped with 10 μl DNase Incubation mix, incubated for 5 minutes at room temperature, and centrifuged at 10,000 g for 90 seconds.

RNA in the supernatant was made into 100 μl with water as needed and cleaned using the OneStep PCR Inhibitor Removal Kit (Zymo Catalog No. D6030). Zymo spin columns were prepared with 600 μl of preparation buffer followed by 400 μl and 100 μl of water, all centrifuged at 8000 g for 3 minutes. The sample was then passed through the column by centrifugation at 8000 g for 3 minutes. The sample was then cleaned a second time using reagents from the RNeasy MinElute Cleanup kit (Qiagen Catalog No. 74204). RNA samples were mixed with 350 μl RLT buffer and 900 μl EtOH and loaded onto the RNeasy MinElute column by centrifugation at> 8,000 g. The column was washed with 500 μl of RPE wash buffer followed by 500 μl of 80% ethanol and then dried as recommended by the manufacturer. For elution, 15 μl of water was added to the column, incubated for 1 minute at room temperature, and then collected in a microcentrifuge tube by centrifugation at 16,000 g for 1 minute. For quality control, 1 μl (out of 15 μl) was analyzed using RNA 6000 Pico Reagent (Agilent) with a bioanalyzer.

CDNA was synthesized using SuperScript IV reverse transcriptase (Life Technologies Catalog No. 18090050) and then treated with NEBNext® Second Strand Synthesis Kit (New England BioLabs) Catalog Number E6. RNA (up to 10 μl) was mixed with 1.12 μl of random hexamer primer (3 mg / ml) and 0.56 μl of dNTP (10 mM each) in a total volume of 14 μl, incubated for 5 minutes at 65 ° C., then 4 ° C. Cooled to. The sample was then mixed with 0.43 μl water, 4 μl SSIV buffer, 0.57 μl DTT (0.1 M) and 1 μl reverse transcriptase (200 U / μl) at 23 ° C. for 10 minutes at 50 ° C. It was incubated for 50 minutes at 80 ° C. for 10 minutes and then kept at 4 ° C. For second chain synthesis, the reaction was supplemented with 4 μl of reaction buffer and 2 μl of NEBNext enzyme to a total volume of 40 μl with water and incubated for 1 hour at 16 ° C. The dsDNA was cleaned with AMPure XP SPRI beads (Beckman Coulter Inc. Catalog No. A63882). 40 μl of dsDNA was mixed with 40 μl of Low EDTA TE (Swift Biosciences Catalog No. 90296) and 144 μl of SPRI beads for 2 minutes and then incubated for 3 minutes at room temperature. Beads were collected using a magnetic rack, washed twice with 200 μl of 80% ethanol and air dried for 5 minutes.

Reagents from Accel-NGS 2S Plus DNA Library Kit (Swift Biosciences Catalog No. SP-2014-96), and unique dual indexes (Unique Dual Indexes) (UDI) (Integrated DNA Techniques). SPRI beads were suspended in 53 μl Low EDTA TE, 6 μl buffer W1 and 1 μl enzyme W2 and incubated for 10 minutes at 37 ° C. 108 μl of PEG NaCl solution (Swift Biosciences Catalog No. 90196) was added. The beads were mixed for 2 minutes, incubated for 3 minutes at room temperature, and then collected in a magnetic rack for 5 minutes. After removing the supernatant, the beads were washed twice with 180 μl of 80% ethanol for 30 seconds and then air dried. The beads were resuspended in 30 μl Low EDTA TE, 5 μl buffer G1, 13 μl reagent G2, 1 μl enzyme G3 and 1 μl enzyme G4 and incubated at 20 ° C. for 20 minutes. 82.5 μl of PEG NaCl solution was added, then mixed for 2 minutes, incubated for 3 minutes at room temperature, and recovered in a magnet rack for 5 minutes. After removing the supernatant, the beads were washed twice with 180 μl of 80% ethanol for 30 seconds and then air dried for 1 minute. The beads were resuspended in 20 μl Low EDTA TE, 5 μl reagent Y2, 3 μl buffer Y1 and 2 μl enzyme Y3 and incubated for 15 minutes at 25 ° C. 49.5 μl of PEG NaCl solution was added, then mixed for 2 minutes, incubated for 3 minutes at room temperature and recovered in a magnet rack for 5 minutes. After removing the supernatant, the beads were washed twice with 180 μl of 80% ethanol for 30 seconds and then air dried for 1 minute. The beads are resuspended in 30 μl Low EDTA TE, 5 μl buffer B1, 2 μl reagent B2, 9 μl reagent B3, 1 μl enzyme B4, 2 μl enzyme B5 and 1 μl enzyme B6, and incubated at 40 ° C. for 10 minutes. Then, the temperature was returned to 25 ° C. 70 μl of PEG NaCl solution was added, then mixed for 2 minutes, incubated for 3 minutes at room temperature, and recovered in a magnet rack for 5 minutes. After removing the supernatant, the beads were washed twice with 180 μl of 80% ethanol for 30 seconds and then air dried for 1 minute. The beads were resuspended in 21 μl Low EDTA TE by mixing for 2 minutes followed by incubation for 2 minutes. The beads are collected in a magnetic rack, the supernatant is transferred to a new plate, 5 μl Illumina UDI Primer Mix (1-72) (Integrated DNA Technologies), 10 μl Low EDTA TE, 4 μl reagent R2, 10 μl buffer R3 and It was mixed with 1 μl of enzyme R4. The PCR reaction was heated to 98 ° C. for 30 seconds, cycled 16 times at 98 ° C. for 10 seconds, 60 ° C. for 30 seconds and 68 ° C. for 60 seconds, and then held at 4 ° C. 70 μl of SPRI beads were added. The beads and sample were mixed for 2 minutes, incubated for an additional 2 minutes and collected in a magnet rack for 5 minutes. After removing the supernatant, the beads were washed twice with 180 μl of 80% ethanol for 30 seconds and then air dried for 1 minute. The nucleic acid was eluted with 21 μl of water.

Cf-mRNA sequencing library by enriching cDNA and ERCC DNA using the Sure Select XT V6 whole exome + UTR capture probe and ERCC capture probe in connection with the SureSelect Custom Reagent kit (Agilent Technologies Catalog No. 931170). Formed. Up to 10 indexed samples with a total cDNA library mass of 750-1000 ng were pooled. Vacuum centrifugation was used to reduce the volume to 3.4 μl. The sample was then mixed with 5.6 μl of SureSelect XT2 Block Mix. A 9 μl sample was transferred to a PCR strip tube, sealed, incubated at 95 ° C. for 5 minutes, and then held at 65 ° C. for at least 5 minutes. 1.5 μl water, 0.5 μl SureSelect RNase Block, 6.63 μl Hyb1, 0.27 μl Hyb2, 2.65 μl Hyb3, 3.45 μl Hyb4, 1 μl ERCC Capture Library (Agilent) and 5 μl. Capture Library ≧ 3Mb (all Exon V6 60Mb) was added and the samples were incubated overnight at 65 ° C. with a heating lid. MyOne streptavidin beads (50 μl) were prepared by 4 washes with 200 μl of SureSelect binding buffer. The pooled sample was added to the streptavidin beads and mixed for 30 minutes at 1800 rpm. The beads were collected with a magnet and washed with 200 μl of SureSelect wash buffer 1 for 15 minutes and then with 200 μl of SureSelect wash buffer 2 for 10 minutes at 65 ° C. three times. Nucleic acid was eluted from the beads by incubation in 20 μl water at 95 ° C. for 5 minutes, transferred to new tubes and mixed with 6 μl water, 25 μl 2X Herculase Master Mix and 1 μl XT2 Primer Mix. The sample was incubated at 98 ° C. for 2 minutes, cycled 15 times at 98 ° C. for 30 seconds, 60 ° C. for 30 seconds and 72 ° C. for 1 minute, stretched at 72 ° C. for 10 minutes, and then held at 4 ° C. .. The reaction was cleaned with 90 μl of AMPureXP beads and eluted with 15 μl of water. The products were analyzed by Kapa qPCR and capillary electrophoresis. Dilutions were prepared at 10 mM Tris-HCl, pH 8 for Kap a qPCR. Capillary electrophoresis was performed on a bioanalyzer.

After quantification, the sequencing pools were denatured and diluted according to their size and according to Illumina's recommendations to achieve optimal cluster formation. A PhiX control was added to the sample as a reference. Using a 1000 uL pipette, all dilution libraries were loaded into reservoir # 10 according to the instructions for NextSeq 500 (Illumina). Sequencing trials were performed using Illumina Basespace according to their instructions. Sequencing was done on a paired end and the read cycle was set to 76. NextSeq was selected as the sequencing machine. Sequencing trials were initiated on the NextSeq 500 according to the manufacturer's instructions.

Base calls were made using FASTQ Generation Application on the BaseSpace platform (Illumina Inc). For sequencing data analysis, cutadapt (v1.11) was used to remove adapter sequences and trim low quality bases. Reads shorter than 15 base pairs after trimming were excluded from subsequent analysis. Using STAR (v2.5.2b) and the GENCODE v24 gene model, read sequences larger than 15 base pairs were aligned with the human reference genome GRCh38. Duplicate reads were removed using the samtools (v1.3.1) rmdup command.

For cell-type deconvolution, normalization was performed in which the expression level of each gene was divided by its maximum over all samples. This step rescales expression levels between different genes to avoid control of the degradation process by a small number of highly expressed genes. The normalized expression matrix is then presented in scikit-learn in the Python library Cykit Learn. Decomposition. It was subjected to non-negative matrix factorization (NMF) using NMF. NMF degradation achieves a more concise representation of the data, X = WH, by decomposing the expression matrix into the product of two matrices, where X in the equation is n rows (n samples) and m columns. It is an expression matrix having (m genes), W is a coefficient matrix having n rows (n samples) and p columns (p components), and H is a p row (p components). ) And m columns (m genes). W is, in a sense, a summary of the original matrix H with a reduced number of dimensions. H contains information on how much each gene contributes to the component. A biological interpretation of the derived components was achieved by performing pathway analysis on the top genes that contribute most to each component.

表１０：無細胞ｍＲＮＡにおいて検出された低ストリンジェンシー非血液遺伝子

Whole blood and matched plasma samples were sequenced to identify "non-blood" genes. A gene is considered "non-blood" if its normalized expression (transcriptions per million, TPM) is three-fold higher in plasma than whole blood (containing blood cells). The "non-blood" gene is probably derived from tissues and / or organs rather than blood cells. A blood cell polynucleotide has a sequence aligned with a blood cell gene and is not a non-blood polynucleotide having a sequence aligned with a non-blood gene. Non-blood genes ranged from 11% to 24%, representing 18% of the TPM in the library on average. Non-blood genes corresponded to 15% of all detected genes (genes detected and counted when TPM ≧ 3), ranging from 8% to 24%. A list of 2,855 non-blood genes detected in this study is presented in Table 1. A list of non-blood genes with lower stringency detected in this study is presented in Table 10.
Table 1: Non-blood genes detected in cell-free mRNA

Table 10: Low stringency non-blood genes detected in cell-free mRNA

(Example 2 Concentration of tissue-derived cf-mRNA by size fractionation)
Generally, most RNA in blood is found in blood cells. Methods for preparing serum and plasma generally involve slow rotation to remove most blood cells. However, residual blood cells are a major source of noise that can interfere with the analysis of cf-RNA.

表３：無細胞ｍＲＮＡにおいて検出された赤血球特異的遺伝子（２３）

表４：無細胞ｍＲＮＡにおいて検出された血小板特異的遺伝子（３２６）

表５：無細胞ｍＲＮＡにおいて検出された好中球特異的遺伝子（２３９）

表６：無細胞ｍＲＮＡにおいて検出された脳特異的遺伝子（１６３）

表７：無細胞ｍＲＮＡにおいて検出された肝臓特異的遺伝子（６３）

The GTEx tissue expression database was used to identify tissue-specific and organ-specific genes. A gene is considered tissue or organ specific if its expression is at least 5-fold higher in one tissue or organ compared to all other tissues and organs. The tissue-specific and organ-specific genes detected in this study are presented in Tables 2-7.
Table 2: Tissue-specific genes detected in cell-free mRNA (455)

Table 3: Erythrocyte-specific genes detected in cell-free mRNA (23)

Table 4: Platelet-specific genes detected in cell-free mRNA (326)

Table 5: Neutrophil-specific genes detected in cell-free mRNA (239)

Table 6: Brain-specific genes detected in cell-free mRNA (163)

Table 7: Liver-specific genes detected in cell-free mRNA (63)

表９：無細胞ｍＲＮＡにおいて検出された妊娠関連遺伝子

Tables 8 and 9 present genes of interest for the diagnosis of liver-specific diseases and pregnancies that do not meet the stringent criteria for non-blood genes.
Table 8: Additional liver-specific genes detected in cell-free mRNA

Table 9: Pregnancy-related genes detected in cell-free mRNA

(Example 2 Concentration of tissue-derived cf-mRNA by size fractionation)
Generally, most RNA in blood is found in blood cells. Methods for preparing serum and plasma generally involve slow rotation to remove most blood cells. However, residual blood cells are a major source of noise that can interfere with the analysis of cf-RNA.

Size selection made for serum or plasma can increase the ratio of solid tissue-derived cf-mRNA compared to blood cell-derived cf-mRNA. To prepare plasma or serum, cells are settled at a rotation of 1,600 g. A second centrifugation step was performed to concentrate tissue-derived cf-mRNA. Plasma was centrifuged for 10 minutes at various rates producing sedimentation forces ranging from 1,900 g to 16,000 g, followed by cf-RNA isolation, cDNA synthesis, library preparation and sequencing. As the rate of centrifugation increases, platelets, which are RNA transcripts from blood cell components, and neutrophil transcripts (representing transcripts from blood cells) are tissue-specific, such as transcripts from the liver or brain. It decreased more rapidly than the transcript, resulting in an increased ratio of non-blood cf-mRNA to blood cell-derived cf-mRNA (FIGS. 2A-3B). This enrichment was offset by a decrease in the number of detectable tissue-derived genes (Fig. 4). Optimal rates for the preparation of low-noise, but representative and diverse cf-mRNA libraries depend on application and are often in the range of 10,000 g to 16,000 g. For example, faster centrifugation rates are preferred for analysis of liver cf-mRNA transcripts as compared to brain cf-mRNA transcripts. 16,000 g of g was used for the results presented below.

Size selection was also performed by membrane filtration with a size cutoff of 0.8 μm, 0.45 μm or 0.2 μm (FIG. 2). When the pore size decreases, platelets, which are RNA transcripts from blood cell components, and neutrophil transcripts (representing transcripts from blood cells) are tissue-specific transcripts such as transcripts from the liver or brain. It decreased more rapidly than the substance, resulting in an increase in the ratio of non-blood cf-mRNA to blood cell-derived cf-mRNA. (Fig. 2).

(Example 3 Selection of cf-mRNA extraction methodology)
To optimize cf-mRNA extraction, phenol-based extraction of total cf-RNA: TRIzol, miRNeasy (Qiagen), Direct-zol (Zymo Research), nucleic acid ZOL (Machery-Nagel), mirVana (Life) Dissolution following a technique based on external vesicle capture (either based or non-phenolic): exoRNAsy (Qiagen), ExoComplete (Hitachi); nucleic acid extraction following vesicle immunoselection or immunodepletion; Total RNA / nucleic acid isolation following lysis: Plasma / Serum RNA Purification Mini Kit (Norgen), QIAamp Circulating Nucleic Acids kit (“CNA kit”), various QIAamp cfDNA / RNA kits (Qiagen, etc.) The kit and method were evaluated. The CNA kit was selected because it showed the best balance between efficiency, scalability, linearity and consistency of cf-RNA extraction (see Figure 5). The CNA kit is a total cf-RNA extraction kit regardless of whether the circulating cf-RNA is transferred as free RNA or protected by proteins, lipids or vesicles.

The cf-RNA tends to be degraded in vivo and is further fragmented during the extraction process. MiRNAs are also typically shorter than mRNAs. Therefore, Qiagen's "miRNA purification" protocol was selected in place of their standard "nucleic acid purification" protocol.

Several changes were made to the protocol supplied with the CNA kit to maximize extraction efficiency and consistency. Do not add the protocol, (1) carrier RNA, to the lysis buffer as it interferes with the sequencing results; (2) spike in ERCC external reference RNA during lysis; (3) lyse buffer. Pre-warming to a dissolution temperature (60 ° C instead of 25 ° C); (4) extending the dissolution time from 30 minutes to a minimum of 45 minutes; (5) a second to better remove the inhibitor from the sample. Adapted by adding a 100% ethanol wash step; and (6) adding a second nucleic acid lysis step to more completely remove RNA from the column. The size distribution of the polynucleotides extracted by the improved method demonstrated improved yields of fragmented cf-RNA compared to the standard nucleic acid purification protocol (FIGS. 6A-6B). This improved extraction method with the CNA kit yielded more cf-mRNA than the QIAamp cfDNA / RNA kit and showed linearly better results with increasing or decreasing plasma input (FIG. 7).

A dedicated enzymatic DNase step was incorporated into the protocol to remove DNA contamination and carryover. Low-level DNA contamination can cause errors in quantifying gene expression, but it may also be valid for cf-RNA isolation due to the very low amount of cf-RNA in serum or plasma. Some commercially available cf-RNA extraction kits either ignore the step of removing DNA (eg, many phenol-based kits) or may not be optimal for complete removal of DNA. I recommended processing. In fact, DNase I can be sensitive to salts that are abundant in RNA extraction and can have low efficiency at low DNA concentrations (this may be the case when working with cell-free body fluids). .. A mutant version of DNase I with increased affinity for DNA, Turbo DNase (Ambion), was used. This is because it is particularly effective in removing trace amounts of DNA and is more resistant to inhibitors. DNase treatment was performed according to the Ambion protocol, except that the amount of enzyme was increased to 2.5-3 μl per sample. DNase I treatment eliminated a substantial amount of contaminating nucleic acid that would otherwise interfere with cf-RNA analysis (FIG. 8).

Titration of the amount of the extracted material into the downstream reaction revealed variable trace inhibitors from the blood that reduced the efficiency of the biochemical reaction. Therefore, inhibitor removal steps and silica membrane-based purification steps were performed after DNAse treatment. Inhibitor removal was performed using the OneStep PCR Inhibitor Removal Kit (Zymo) and additional column washes to ensure complete removal of preparation buffer. Cleanup for this column increases the apparent yield of cf-RNA by removing impurities that interfere with the enzyme, and the OneStep column yields a higher apparent yield than the Micro Bio-Spin column (Bio-Rad). Had (Fig. 9). Cleanup using the OneStep PCR Inhibitor Removal kit also preserved the recovery of fragmented cf-RNA polynucleotides (FIGS. 10A-10B). Silica membrane purification was performed using the MinElute PCR Purification Kit (Qiagen) with higher ethanol for maximum recovery.

The final cf-RNA extraction and purification process dramatically reduces the frequency of assay failures due to suboptimal yields of RNA below 20 pg, and cell-free mRNA using a range of input volumes (100 ul-3 ml). Produced an efficient and linear recovery of (Figs. 11-12).

(Example 4 cDNA synthesis, library preparation, and total exosome capture)
Commercially available low input RNA sequencing kits generally include reagents for cDNA synthesis and removal of RNA species of no informative value. The cDNA synthesis step in SMARTer (Takara) has been found to be inefficient. Therefore, a three-step process using a dedicated first cDNA synthesis step and a second-strand synthesis reaction followed by a commercially available kit optimized for library preparation from low levels of dsDNA and subsequent capture of whole exosomes. Developed a strategy.

SuperScript IV (Invitrogen) was selected as the enzyme for reverse transcription. Because it uses cf-RNA input compared to iScript (Bio-Rad), qScript (Quantavio), SuperScript III (Invitrogen) and SMARTScribe (Takara), for enzyme efficiency, linearity and trace inhibitors. This is because it demonstrated increased resistance. The conversion efficiency in SuperScript IV is (1) primed with random hexamers instead of oligo dT, (2) the primer concentration is increased 30-fold to 3 mg / ml, and (3) reaction in case of emergency. It was further optimized by extending the time from 10 minutes to 50 minutes. The optimized SuperScript IV method yielded more cDNA than iScript (FIG. 13) and had better linearity than SMARTScribe (FIG. 14).

A double-stranded cDNA was produced by a double-strand synthesis reaction using the NEBNext enzyme. No cleanup was performed between the first and second chain synthesis. The second chain synthesis reaction was optimized by reducing the reagents used and the total reaction volume by 50%.

Accel-NGS 2S Plus is sequenced from Accel-NGS 1S Plus (Swift Biosciences) and other, eg, NxSeq® UltraLow DNA Library Preparation Kit (Lucien), in small amounts. It turned out to be the most robust and scalable library preparation method. Specifically, the number of unique sequence fragments was approximately 30% higher with Accel-NGS 2S Plus compared with Accel-NGS 1S Plus (FIG. 15A). The reagents remaining from the NEBNext 2nd chain synthesis step did not match the chemistry of the Repair I step of Accel-NGS 2S Plus. Therefore, the cDNA was cleaned using 1.8x SPRI beads prior to Repair I. Repair I was performed with SPRI beads in solution to minimize sample loss, and polyethylene glycol (PEG) was added after repair I to promote DNA binding to the beads. To maximize recovery while avoiding contaminants such as aptamers and dimers, increase the amount of beads 1.8 times for Repair I, 1.65 for Repair II, and 1.65 for Ligation I. And ligation II was adjusted to 1.4. These improvements to the Accel-NGS 2S Plus protocol increased the number of unique sequence fragments by approximately 20% (FIG. 15B).

Both ends of each library preparation were uniquely labeled with UDI during PCR and prior to enrichment. I chose UDI instead of the standard index to minimize index hopping. This was particularly important for the cf-RNA library given the small number of copies of the input material. When using the standard index, the sequencing read was misdesignated as a negative control (NTC). This contamination is reduced by the use of UDI (FIGS. 16A-16B).

cf-mRNA was enriched from total cf-RNA by total exosome capture. This method was chosen over rRNA depletion because mRNA constitutes less than 10% of the circulating RNA molecules. Capture is performed with either an RNA bait or a DNA capture probe (IDT). RNA baits are preferred because of the higher coverage for the specific region of interest. However, both can be used. For normalization and quality control, whole exome probes were combined with another set of probes designed to capture 35 ERCC standards covering a wide range of copy numbers and sizes, and these were spiked during the extraction step. did. Capture was performed using a pool of up to 10 cDNA samples according to an improved version of the Agilent protocol using XT2 blockers and reagents with XT probes. Different enrichment strategies were compared by sequencing the percentage of RNA polynucleotides resulting from mRNA and other sources, such as ribosomal RNA, mitochondrial RNA, non-coding RNA and other RNA species. The mRNA fraction was substantially higher in total exosomal capture compared to negative enrichment due to rRNA depletion and the starting pool of total RNA (FIG. 17). Specifically, approximately 80% of sequence reads from total exon capture material are mRNA sequences, approximately 45% of sequences after rRNA depletion are mRNA sequences, and less than 5% of sequences from total RNA are mRNA sequences. there were.

The cDNA synthesis, library preparation and total exosome capture methods presented in this section were constructed using the SMARTer kit and are sequences that better represent a wide range of cf-mRNA compared to rRNA-depleted libraries. Brought a single lead. The sensitivity to detect a small amount of spike-in ERCC standard material was improved by 5 to 20 times (Fig. 18A). This increased sensitivity facilitated the detection of substantially more protein-encoding genes (Fig. 18B). Using a known concentration of spike-in ERCC standard material, the detection sensitivity was estimated to be approximately 14 copies (FIG. 18C).

(Example 5 Comparison with other cf-mRNA libraries)
The cell-free mRNA library prepared by the method of Example 1 was superior to the "Pan et al." Cell-free mRNA library (Pan et al, Clin Chem. 2017 Nov; 63 (11): 1695-1704). For this analysis, raw sequencing data from both studies were processed using the bioinformatics pipeline described in Example 1. The Pan library was prepared from an equivalent of 500 μl serum per preparation, but only 165 μl serum per preparation was used to prepare the Example 1 library. Despite approximately half the sequencing leads per preparation (FIG. 19A), the Example 1 protocol (improved using centrifugation at 16,000 g and concentration with a DNA capture probe) is approximately. 6-fold more unique fragments (Fig. 19B), approximately 3-fold more protein-encoding genes (Fig. 19C), approximately 4-fold more genes with> 80% coverage (Fig. 19D), and approximately 8-fold more liver genes (Fig. 19E). ) Was brought.

Preferred embodiments of the present disclosure have been shown and described herein, but it will be apparent to those of skill in the art that such embodiments are provided by way of example only. Numerous variants, modifications and substitutions will now come to the minds of those skilled in the art without departing from the present disclosure. It should be understood that various alternatives of the embodiments described herein are available in the practice of the present disclosure. The following claims define the scope of the present disclosure, and it is intended that the methods and structures within these claims and their equivalents are included in these claims.

Claims (86)

A method for preparing a cf-RNA sample.
It comprises (a) centrifuging the biological sample at 1,600 g to 16,000 g and (b) isolating RNA from the biological sample.
At least 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 non-blood genes selected from the list in Table 1 or low stringency non-blood genes selected from Table 10 are: The method present in the cf-RNA sample. The method according to claim 1, wherein the biological sample is cell-free. The method according to claim 1 or 2, wherein the biological sample is serum, plasma, saliva, urine, interstitial fluid, cerebrospinal fluid, semen, vaginal fluid, amniotic fluid, tears, synovial fluid, mucus or lymph. The method according to any one of claims 1 to 3, wherein the biological sample is serum or plasma. The method according to any one of claims 1 to 4, further comprising a step of performing size selection or immunoselection in the biological sample prior to (b). 5. The method of claim 5, wherein the step of performing the size selection comprises centrifuging the biological sample. The method of claim 6, wherein the centrifugation is performed for at least 1 minute, at least 10 minutes, 5 minutes to 20 minutes, 10 minutes to 15 minutes, or about 10 minutes. 5. The method of claim 5, wherein the step of performing the size selection comprises filtering the sample. The method according to any one of claims 1 to 8, wherein the biological sample is centrifuged at 10,000 g to 15,000 g. The method according to any one of claims 1 to 9, wherein the biological sample is centrifuged at about 12,000 g. The method according to any one of claims 1 to 10, wherein (b) comprises isolating the extracellular vesicle from the biological sample and isolating the RNA from the extracellular vesicle. .. 11. The method of claim 11, wherein the extracellular vesicles are exosomes. The method according to any one of claims 1 to 12, wherein (b) comprises isolating the nucleoprotein complex from the biological sample and isolating the RNA from the nucleoprotein complex. .. The method according to any one of claims 1 to 13, further comprising treating the RNA with a deoxyribonuclease. 15. The method of claim 14, wherein the deoxyribonuclease is TurboDNase I. The method of claim 14 or 15, wherein the RNA is in solution when treated with the deoxyribonuclease. The method according to any one of claims 1 to 16, wherein (b) comprises contacting the RNA with at least one of an affinity column, a desalted column, or a silica membrane. 17. The method of claim 17, wherein (b) comprises contacting the RNA with an affinity column, a desalting column, and a silica membrane. The method of any one of claims 1-18, further comprising the step of concentrating at least one protein-encoding nucleotide sequence. The method of any one of claims 1-19, comprising the step of depleting the ribosomal RNA sequence from said RNA. A method for identifying cf-RNA molecules,
(A) Steps to isolate RNA from a biological sample,
(B) Steps to prepare a cDNA library from the RNA,
The list in Table 1, comprising (c) sequencing the cDNA library and (d) identifying at least one gene in the cDNA library, wherein the biological sample is substantially cell-free. A method of detecting at least 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 non-blood genes selected from, or low stringency non-blood genes selected from Table 10. .. 21. The method of claim 21, wherein the biological sample is cell-free. (E) The method of claim 21 or 22, further comprising aligning the sequence from the cDNA library with the reference genome. 13. The method described. The method according to any one of claims 21 to 23, wherein the biological sample is serum or plasma. The method of any one of claims 21-25, wherein at least 35, 50, 75, 100, 200, 300, 400 or 500 tissue-specific genes selected from Table 2 are identified. The method of any one of claims 21-25, wherein at least 20, 30, 40, 50, 100 or 150 brain-specific genes selected from Table 6 are identified. One of claims 21 to 25, wherein at least 3, 5, 10, 20 or 50 liver-specific genes selected from Table 7 or one liver diagnostic gene selected from Table 8 are identified. The method described in the section. The method of any one of claims 21-25, wherein at least 3, 5, 10 or 15 pregnancy-related genes selected from Table 9 are identified. 21-28, wherein the first gene is identified and the RNA comprises less than 500, 200, 150, 100, 50, 25 or 15 cf-mRNA polynucleotides aligned with the first gene. The method described in any one of the items. The method of any one of claims 21-30, wherein at least 2, 4, 6, 8 or 10 unique fragments are detected per 100 reads. The method of any one of claims 21-31, wherein at least 2, 4, 6, 8 or 10 protein-encoding genes are detected per 10,000 reads. The method of any one of claims 21-32, further comprising the step of performing size selection or immunoselection in the biological sample prior to (a). 33. The method of claim 33, wherein the step of performing the size selection comprises centrifuging the biological sample. 34. The method of claim 34, further comprising centrifuging the biological sample at 1,600 g to 16,000 g. 35. The method of claim 35, wherein the centrifugation is performed for at least 1 minute, at least 5 minutes, at least 10 minutes, 5 minutes to 20 minutes, 10 minutes to 15 minutes, or about 10 minutes. 35. The method of claim 35 or 36, wherein the biological sample is centrifuged at 10,000 g to 15,000 g. 35. The method of claim 35 or 36, wherein the biological sample is centrifuged at about 12,000 g. 34. The method of claim 34, wherein the step of performing the size selection comprises filtering the sample. The method according to any one of claims 21 to 39, wherein (a) comprises isolating the extracellular vesicle from the biological sample and isolating the RNA from the extracellular vesicle. .. 40. The method of claim 40, wherein the extracellular vesicle is an exosome. The method according to any one of claims 21 to 41, wherein (a) comprises isolating the nucleoprotein complex from the biological sample and isolating the RNA from the nucleoprotein complex. .. The first of the cDNA polynucleotides further comprises the step of adding an exogenous RNA polynucleotide comprising the first nucleotide sequence to the biological sample and the step of detecting a cDNA polynucleotide comprising the first nucleotide sequence. The method of any one of claims 21-42, wherein the nucleotide sequence comprises timine at each position where the first nucleotide sequence of the RNA polynucleotide comprises uracil. The method of any one of claims 21-43, further comprising treating the RNA with a deoxyribonuclease. 44. The method of claim 44, wherein the deoxyribonuclease is TurboDNase I. 44. The method of claim 44 or 45, wherein the RNA is in solution when treated with the deoxyribonuclease. The method of any one of claims 21-46, wherein (a) comprises contacting the RNA with at least one of an affinity column, a desalted column, or a silica membrane. 47. The method of claim 47, wherein (a) comprises contacting the RNA with an affinity column, a desalting column, and a silica membrane. The method of any one of claims 21-48, wherein (b) comprises contacting the RNA with a primer comprising a random sequence. 49. The method of claim 49, wherein the primer is a hexanucleotide. The concentration of the hexanucleotide is at least 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 150 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1000 μM, 1100 μM, 1200 μM, 1300 μM, 1400 μM or 1500 μM. The method according to claim 50. The method according to any one of claims 21 to 51, wherein (b) comprises forming a single-stranded cDNA. 52. The method of claim 52, further comprising contacting the RNA with reverse transcriptase to form the single-stranded cDNA. The method according to any one of claims 51 to 53, further comprising the step of forming a double-stranded cDNA from the single-stranded cDNA. 54. The method of claim 54, further comprising contacting the single-stranded DNA with a NEBNext DNA polymerase to form the double-stranded cDNA. 55. The method of claim 55, further comprising ligating the unique dual index to both ends of the double-stranded cDNA. The method of any one of claims 21-56, further comprising the step of concentrating at least one protein-encoding nucleotide sequence. 57. The method of claim 57, comprising depleting the ribosomal RNA sequence from said RNA. 57. The method of claim 57, comprising depleting the ribosomal RNA sequence from the cDNA library. The method of any one of claims 57-59, comprising the step of isolating the at least one protein coding sequence from said RNA. The method of any one of claims 57-59, comprising the step of isolating the at least one protein coding sequence from the cDNA. 61. The method of claim 61, comprising the step of hybridizing whole exome bait to the cDNA. 62. The method of claim 62, wherein the whole exome bait comprises an RNA polynucleotide. 62. The method of claim 62, wherein the whole exome bait comprises a DNA polynucleotide. A method for detecting at least 10, 20, 30, 50 or 100 non-blood cf-mRNA genes in a biological sample.
(A) A step of centrifuging a serum or plasma sample at 8,000 g to 16,000 g for at least 10 minutes to form a supernatant.
(B) Step of extracting RNA from the supernatant,
(C) The step of contacting the RNA with a deoxyribonuclease,
(D) The step of forming cDNA from the RNA,
(E) A step of preparing a cDNA library from the cDNA,
(F) Steps to sequence the cDNA library, and (g) sequences resulting from at least 10, 20, 30, 50 or 100 non-blood cf-mRNA genes per biological sample aligned with the reference genome. A method that includes the steps to identify. (H) Claim 65 further comprises contacting the cDNA library with a bait containing a polynucleotide fragment from at least 10, 20, 30, 50 or 100 genes of interest to enrich the gene to be translated. The method described. (D) comprises contacting the RNA with reverse transcriptase to form a single-stranded cDNA, and contacting the single-stranded cDNA with a double-stranded synthase to form a double-stranded cDNA. , The method of claim 65. 65. The method of claim 65, further comprising ligating the unique dual index to the cDNA library to form an indexed cDNA library. 28. The method of claim 68, further comprising pooling up to 10 indexed cDNA libraries. 39. The method of claim 69, wherein (f) comprises performing massively parallel sequencing with respect to the pooled cDNA library. The invention according to any one of claims 65 to 70, wherein at least 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 genes are detected in the biological sample. Method. To identify sequences that result from at least 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 non-blood cf-mRNA genes per biological sample. The method of any one of claims 65-71, which is aligned with the reference genome. The method according to any one of claims 65 to 70, further comprising contacting the single-stranded cDNA with a double-stranded synthase to form the double-stranded cDNA. The method according to any one of claims 65 to 70, wherein (c) is carried out in a solution. A method for detecting at least 10, 20, 30, 50 or 100 non-blood cf-mRNA genes in a biological sample.
(A) A step of centrifuging or filtering a serum or plasma sample at 1,900 g to 16,000 g.
(B) Step of extracting RNA sample from supernatant,
(C) The step of contacting the RNA sample with a deoxyribonuclease,
(D) The step of contacting the RNA with reverse transcriptase to form a single-stranded cDNA.
(E) A step of contacting the single-stranded cDNA with a double-stranded synthase to form a double-stranded cDNA.
(F) The step of preparing a cDNA library from the double-stranded cDNA,
(G) A step of contacting a bait containing a polynucleotide fragment with an indexed cDNA library to concentrate the gene to be translated.
(H) Steps to sequence the cDNA library, and (i) Sequences resulting from at least 10, 20, 30, 50 or 100 non-blood cf-mRNA genes per biological sample, aligned with the reference genome. A method that includes the steps to identify. (J) The method of claim 65, further comprising the step of adding a unique dual index to the cDNA to form an indexed cDNA library. (K) The method of claim 66, further comprising pooling up to 10 indexed cDNA libraries. (L) The method of claim 77, further comprising performing massively parallel sequencing on the pooled cDNA library. The method of any one of claims 75-78, wherein (c) is performed in solution. Cf-mRNA containing cDNA molecules derived from at least 500, 600, 700, 800, 900, 1000, 1250 or 1500 non-blood genes selected from the list in Table 1 or low stringency non-blood genes selected from Table 10. Sequencing library. From at least 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 non-blood genes or Table 10 selected from the list in Table 1 per 1,000,000 cDNA polynucleotides. A cf-mRNA sequencing library containing at least one cDNA molecule originating from a low stringency non-blood gene of choice. The cf-mRNA sequencing library of claim 80 or 81, comprising cDNA molecules originating from at least 35, 50, 100, 200, 300, 400 or 500 tissue-specific genes selected from Table 2. The cf-mRNA sequencing library of claim 80 or 81, comprising cDNA molecules originating from at least 18, 20, 50 or 100 brain-specific genes selected from Table 6. The cf-mRNA according to claim 80 or 81, comprising a cDNA molecule derived from at least 3, 5, 10, 20 or 50 liver-specific genes selected from Table 7 or the liver gene of interest selected from Table 8. Sequencing library. The cf-mRNA sequencing library of claim 80 or 81, comprising cDNA molecules originating from at least 3, 5, 10 or 15 pregnancy-related genes selected from Table 9. A cf-mRNA sequencing library containing cDNA polynucleotides originating from at least 2000, 3000, 4000, 5000 or 6000 protein coding genes, wherein at least 8%, 15% or 24% of the protein coding genes are non-blood genes. The cf-mRNA sequencing library.

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